Hypothermic preservation of hepatocytes. III. Effects of resuspension media on viability after up to 7 days of storage

Abstract

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. Using these studies, improved methods of hypothermic liver preservation for transplantation may be developed. In this study, rat hepatocytes were cold-stored for up to 7 days in University of Wisconsin liver preservation solution. At the end of each day of storage hepatocytes were resuspended in Krebs-Henseleit buffer or tissue-culture medium (Liebovitz-15; Fischer's; modified Fischer's, which was similar to Fischer's but with glycine and cysteine added; or Waymouth's medium). Hepatocyte viability was assessed by rewarming and oxygenating the suspensions and measuring the percentage of leakage of lactate dehydrogenase from the cells, the cellular concentration of potassium and the stimulation of respiration by succinate, all measures of plasma membrane integrity. Additionally, concentrations of ATP and glutathione after rewarming and reoxygenation in the various resuspension media were measured. Hepatocyte permeability to lactate dehydrogenase did not increase during cold storage of 1 to 7 days (7.2% +/- 2% leakage), indicating that most of the hepatocytes remained viable during cold storage. However, when rewarmed, loss of viability (leakage of lactate dehydrogenase) was dependent on the composition of the resuspension media. In Krebs-Henseleit buffer, viability was reduced after 2 and 3 days of storage (lactate dehydrogenase leakage on rewarming = 70% to 90%). Leakage of lactate dehydrogenase was reduced significantly after resuspension in tissue-culture media. After 6 days of storage, lactate dehydrogenase leakage from hepatocytes stored in Liebovitz- 15 or modified Fischer's was only about 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold Storage of Rat Hepatocyte Suspensions for One Week in a Customized Cold Storage Solution – Preservation of Cell Attachment and Metabolism

“…In most studies, cell viability in suspension is assessed directly after cold storage or after a short rewarming phase in suspension [27], [28], [29]; although this parameter is very insensitive, loss of cell viability has been described to occur after 24–120 h. Compared to adherent rat hepatocytes [6], rewarming injury was not very pronounced in cell suspensions in the current study. Furthermore, since the crude cell suspension was used for the experiments, cell death during rewarming is likely to be partly due to cell damage already initiated during the cell isolation process.…”

Preservation of lower extremity spare parts using the University of Wisconsin solution

“…This solution is responsible for the longest period of safe preservation of isolated cells, particularly in pancreatic islets and hepatocytes. 15,16 Animal studies have confirmed the efficacy of using UW solution for preservation of fasciocutaneous flaps. 17,18 Some authors have hypothesized that lactobionate is the key component of the UW solution.…”

Toward the survival and function of xenogeneic hepatocyte grafts

“…A favored approach to the storage of cells is hypothermic preservation 1, 55, 56. Among the advantages of this storage method is that it does not require special equipment for preservation and might allow shipping to remote sites for transplantation.…”